<Summary of Exosome>
An exosome is an extracellular granule secreted by cells. Specifically, the exosome is a vesicle having a diameter of approximately 30 nm to 100 nm that is released from the cell having a vesicle structure formed of a lipid bilayer. It has been known that the exosome is incorporated into another cell different from the cell that has secreted the exosome (hereinafter referred to as a secreting cell) and thereby plays a role in information signaling between the secreting cell and the other cell (Non-Patent Documents 1 and 2).
<Stability of Exosome>
It has been known that a structure of the exosome membrane is generally stable compared to a structure of the cell membrane. For example, the experimental results are described in Non-Patent Document 3 that a substance contained in the exosome is stably maintained even in a case where the exosome is subjected to a freeze-thaw process for 4 times without adding a stabilizer such as saccharide under the condition of temperature of −20° C. On the other hand, it has been known that cells called animal cells generally cannot bear the freeze-thaw process unless the above described stabilizer is used. Accordingly, it is presumed that the exosome membrane has a stable structure compared to that of the animal cells.
In addition, a step of forming the membrane structure is different between the exosome and the cell. Additionally, the structure of the exosome membrane and the structure of the cell membrane are compared and both are different in the surface area of the membrane and the type of a surface protein present on the surface of the membrane. Even considering this circumstance, it is considered that the structure of the exosome membrane is stable compared to the structure of the cell membrane. Because the exosome is not a living cell, the exosome is not the target for a cell-death program by apoptosis that can be applied to general cells.
<Relationship Between Exosome and Disease>
Various reports have been made recently on the correlation between the exosome and various diseases. Particularly, the exosome released from cancer cells is being focused on and various research results have been reported regarding the influence of the exosome on a particular disease, that is, a cancer. For example, it has been reported that in a case where factors inducing angiogenesis which are contained in the exosome released from the cancer cells are incorporated into healthy vascular endothelial cells, neogenesis of blood vessels around tumor tissues is induced by the factors inducing the angiogenesis, which leads to a possibility of progression of cancer metastasis and invasion (Non-Patent Documents 4 to 6).
In addition, regarding the correlation between the exosome and various diseases, it is also becoming clear that information related to a disease is contained in the exosome. For example, it is described in Non-Patent Document 3 that the exosome present in the blood of a cancer patient contains substances that are biomarkers of cancer (proteins such as PSA, HER2 and EGFR, microRNA such as miR-21 and miR-200a, messenger RNA such as Apbb1ip and ASPN, DNA such as KRAS, various metabolites, or the like).
Particularly, a microRNA contained in the exosome as a substance that is a biomarker of cancer is considered promising as an examination index of cancer. A database of microRNA in living organisms has already been made in miRBase (URL: http://www.mirbase.org/). Among these, 2588 species of human microRNAs are known as of 2015. Furthermore, it has been known that expression profiles in the above described human microRNAs are different depending on the type of the organs and are also different between normal cells and cancer cells. Considering this circumstance, it has been extensively examined to develop techniques for diagnosing the site and progress degree of cancer by using a blood test analyzing microRNA contained in the exosome in blood (Non-Patent Document 7).
<Method for Isolating and Collecting (Collection) and Removing Exosome>
A technique focusing on a method for isolating and collecting (collection) and removing exosomes is as follows.
As the method for collecting the exosome from body fluids of human (blood, saliva, urine and the like) and a medium of cell cultures, an ultracentrifugation method described in Non-Patent Document 8 and the like is the most generally used technique as of 2015. For example, in a case of collecting the exosome from a cell culture supernatant or body fluids, it is possible to remove large contaminants such as cell fragments using a filter and the like, and then perform the ultracentrifugation under the conditions at 4° C. and 100,000 G for 70 minutes to collect the desired exosome. In addition, Non-Patent Document 8 describes a method for collecting an exosome by precipitating the exosome in a cell culture supernatant or body fluids using a co-precipitating agent such as PEG (Non-Patent Document 8).
In addition, Patent Document 1 discloses a technique of adsorptive removal of an exosome by using an antibody that specifically binds to the exosome. Specifically, Patent Document 1 describes a method for adsorptive removal of an exosome by using an antibody that specifically binds to four-pass transmembrane proteins such as CD9, CD63 and CD81 which are present on the surface of a vesicle of the exosome.
Patent Document 2 describes a method for using an antibody that specifically adsorbs to exosomes derived from cancer cells in order to adsorb and remove the exosomes derived from cancer cells secreted by cancer cells.
Non-Patent Documents 9 and 10 disclose a method for destroying and removing an exosome itself by chemically treating a cell membrane of the exosome. Specifically, Non-Patent Document 9 describes a method for using a surfactant and Non-Patent Document 10 describes a method for using phenol, guanidine, and a surfactant.